QUANTITATIVE POLYMERASE CHAIN-REACTION FOR HEPATITIS-B VIRUS-DNA

To monitor and compare the effect of drugs on hepatitis B virus (HBV) replication in hepatitis patients, an accurate quantitative method wit h high sensitivity is needed. Polymerase chain reaction (PCR) is the m ost sensitive method for the detection of HBV DNA, but because of samp le to sample variability in amplification efficiency, the quantificati on of target nucleic acids by conventional PCR is inaccurate. Therefor e, we developed a competitive PCR method with an internal standard whi ch uses the same primers as the target HBV DNA. The standard was gener ated from the HBV S gene by PCR site-directed mutagenesis and differed from the native S gene by a single base which introduced an internal restriction enzyme site. For practical application, this method was us ed to quantitate the effect of the carbocyclic analogue 2'-deoxyguanos ine (2'-CDG) on HBV DNA replication in 2.2.15 cells, an HBV transfecte d HepG2 cell line. A decrease of HBV DNA level in the attomole range w as demonstrated by the assay in the media of 2.2.15 cells treated with 2'-CDG. The results were verified by conventional HBV DNA slot blot a nalysis followed by densitometric scanning. Competitive PCR proved to be a sensitive, accurate and reliable method for HBV quantitation.